Development and Validation of a Pan-Genotypic Real-Time Quantitative Reverse Transcription-PCR Assay To Detect Canine Distemper Virus and Phocine Distemper Virus in Domestic Animals and Wildlife.

Canine distemper virus (CDV) is an animal morbillivirus belonging to the family Paramyxoviridae and has caused major epizootics with high mortality levels in susceptible wildlife species. In recent years, the documented genetic diversity of CDV has expanded, with new genotypes identified in India, the Caspian Sea, and North America. However, no quantitative real-time PCR (RT-qPCR) that has been validated for the detection of all genotypes of CDV is currently available. We have therefore established and characterized a pan-genotypic probe-based RT-qPCR assay based on the detection of a conserved region of the phosphoprotein (P) gene of CDV. This assay has been validated using virus strains representative of six genotypes of CDV in different sample types, including frozen tissue, formalin-fixed paraffin-embedded tissue sections, and virus isolates. The primers and probe target sequences were sufficiently conserved to also enable detection of the phocine distemper virus strains responsible for epizootics in harbor seals in the North Sea in 1988 and 2002. Comparison with two recently published RT-qPCR assays for CDV showed that under equivalent conditions the primers and probe set reported in this study were more sensitive in detecting nucleic acids from an Asia-4 genotype, which displays sequence variation in primer and probe binding sites. In summary, this validated new pan-genotypic RT-qPCR assay will facilitate screening of suspected distemper cases caused by novel genotypes for which full genome sequences are unavailable and have utility in detecting multiple CDV strains in geographical regions where multiple genotypes cocirculate in wildlife.

Longitudinal analysis of pinnipeds in the northwest Atlantic provides insights on endemic circulation of phocine distemper virus.

Phocine distemper virus (PDV) is a morbillivirus that circulates within pinnipeds in the North Atlantic. PDV has caused two known unusual mortality events (UMEs) in western Europe (1988, 2002), and two UMEs in the northwest Atlantic (2006, 2018). Infrequent cross-species transmission and waning immunity are believed to contribute to periodic outbreaks with high mortality in western Europe. The viral ecology of PDV in the northwest Atlantic is less well defined and outbreaks have exhibited lower mortality than those in western Europe. This study sought to understand the molecular and ecological processes underlying PDV infection in eastern North America. We provide phylogenetic evidence that PDV was introduced into northwest Atlantic pinnipeds by a single lineage and is now endemic in local populations. Serological and viral screening of pinniped surveillance samples from 2006 onward suggest there is continued circulation of PDV outside of UMEs among multiple species with and without clinical signs. We report six full genome sequences and nine partial sequences derived from harbour and grey seals in the northwest Atlantic from 2011 through 2018, including a possible regional variant. Work presented here provides a framework towards greater understanding of how recovering populations and shifting species may impact disease transmission.

Canine and Phocine Distemper Viruses: Global Spread and Genetic Basis of Jumping Species Barriers.

Canine distemper virus (CDV) and phocine distemper (PDV) are closely-related members of the Paramyxoviridae family, genus morbillivirus, in the order Mononegavirales. CDV has a broad host range among carnivores. PDV is thought to be derived from CDV through contact between terrestrial carnivores and seals. PDV has caused extensive mortality in Atlantic seals and other marine mammals, and more recently has spread to the North Pacific Ocean. CDV also infects marine carnivores, and there is evidence of morbillivirus infection of seals and other species in Antarctica. Recently, CDV has spread to felines and other wildlife species in the Serengeti and South Africa. Some CDV vaccines may also have caused wildlife disease. Changes in the virus haemagglutinin (H) protein, particularly the signaling lymphocyte activation molecule (SLAM) receptor binding site, correlate with adaptation to non-canine hosts. Differences in the phosphoprotein (P) gene sequences between disease and non-disease causing CDV strains may relate to pathogenicity in domestic dogs and wildlife. Of most concern are reports of CDV infection and disease in non-human primates raising the possibility of zoonosis. In this article we review the global occurrence of CDV and PDV, and present both historical and genetic information relating to these viruses crossing species barriers.

Phocine distemper virus: current knowledge and future directions.

Phocine distemper virus (PDV) was first recognized in 1988 following a massive epidemic in harbor and grey seals in north-western Europe. Since then, the epidemiology of infection in North Atlantic and Arctic pinnipeds has been investigated. In the western North Atlantic endemic infection in harp and grey seals predates the European epidemic, with relatively small, localized mortality events occurring primarily in harbor seals. By contrast, PDV seems not to have become established in European harbor seals following the 1988 epidemic and a second event of similar magnitude and extent occurred in 2002. PDV is a distinct species within the Morbillivirus genus with minor sequence variation between outbreaks over time. There is now mounting evidence of PDV-like viruses in the North Pacific/Western Arctic with serological and molecular evidence of infection in pinnipeds and sea otters. However, despite the absence of associated mortality in the region, there is concern that the virus may infect the large Pacific harbor seal and northern elephant seal populations or the endangered Hawaiian monk seals. Here, we review the current state of knowledge on PDV with particular focus on developments in diagnostics, pathogenesis, immune response, vaccine development, phylogenetics and modeling over the past 20 years.

Phocine distemper virus in seals, east coast, United States, 2006.

In 2006 and 2007, elevated numbers of deaths among seals, constituting an unusual mortality event, occurred off the coasts of Maine and Massachusetts, United States. We isolated a virus from seal tissue and confirmed it as phocine distemper virus (PDV). We compared the viral hemagglutinin, phosphoprotein, and fusion (F) and matrix (M) protein gene sequences with those of viruses from the 1988 and 2002 PDV epizootics. The virus showed highest similarity with a PDV 1988 Netherlands virus, which raises the possibility that the 2006 isolate from the United States might have emerged independently from 2002 PDVs and that multiple lineages of PDV might be circulating among enzootically infected North American seals. Evidence from comparison of sequences derived from different tissues suggested that mutations in the F and M genes occur in brain tissue that are not present in lung, liver, or blood, which suggests virus persistence in the central nervous system.

Viral protein expression and phenotyping of inflammatory responses in the central nervous system of phocine distemper virus-infected harbor seals (Phoca vitulina).

The central nervous system (CNS) represents an important target organ of the phocine distemper virus (PDV). The aim of the present study was to characterize pathological changes in the CNS of harbor seals suffering from natural PDV-infection. The distribution of virus protein and mRNA was investigated by immunohistochemistry (IHC) and in situ hybridization, respectively. In addition, inflammatory and glial cells were characterized by IHC. Polioencephalitis with glial activation, neuronal death and perivascular mononuclear infiltrations in the cerebral cortex was the main histopathological finding. Inflammatory responses, dominated by CD3(+) T-cells and activated microglia/macrophages were associated with a prominent MHC-II upregulation within the CNS. Viral protein was found predominantly in neurofilament-expressing neurons within inflamed areas as demonstrated by immunohistochemical double-labeling. Morbillivirus nucleo-, phospho-, matrix-, fusion- and hemagglutinin-proteins were found in CNS-lesions. The expressions of viral matrix- and fusion-proteins were reduced in severely inflamed plaques. Comparison of viral protein and mRNA expression revealed a diminished amount of viral phosphoprotein preferentially associated with perivascular inflammation. In summary, CNS-lesions in PDV-infected seals are similar to canine distemper virus-induced acute polioencephalitis in dogs and measles virus inclusion body polioencephalitis in men, respectively.

Phocine distemper virus in northern sea otters in the Pacific Ocean, Alaska, USA.

Phocine distemper virus (PDV) has caused 2 epidemics in harbor seals in the Atlantic Ocean but had never been identified in any Pacific Ocean species. We found that northern sea otters in Alaska are infected with PDV, which has created a disease threat to several sympatric and decreasing Pacific marine mammals.

Genetic diversity and phylogenetic analysis of the attachment glycoprotein of phocine distemper viruses of the 2002 and 1988 epizootics.

To investigate the possible origin and spread of the dramatic re-emergent 2002 distemper epizootic observed among seals in Danish Waters, we have sequenced wild-type genes of the attachment (H) glycoproteins of viruses from both the 2002 and 1988 epizootics. Phylogenetic analysis of the H genes of phocine distemper virus (PDV) together with other morbilliviruses, suggests that the re-emergent 2002 PDV is more closely related to a putative recent ancestral PDV than the 1988 PDV isolates. Moreover, upsurges of distemper disease in land-living carnivores linked in time and locality to the 2002 seal epizootic in Danish Waters was investigated and determined to be caused by canine distemper virus, the closest relative of PDV, revealing no direct epidemiological link to the seal epizootics.

Neurological signs in juvenile harbour seals (Phoca vitulina) with fatal phocine distemper.

In 2002, the northern European harbour seal (Phoca vitulina) population experienced an epidemic of phocine distemper virus (PDV) in which 22,000 seals died. Clinical signs were recorded in 20 harbour seal pups admitted to the Seal Rehabilitation and Research Centre with clinical disease, and they were diagnosed PDV infection-positive by RT-PCR postmortem. All 20 had respiratory signs, 14 had conjunctivitis and 10 had neurological signs. Severe neurological signs were one of the criteria for euthanasia during the epidemic, and many pups that were euthanased were not included in this study owing to the lack of complete datasets. Neurological signs were therefore among the most prevalent signs of fatal PDV infection in harbour seal pups. The lymphoid depletion reported in dead seals during the epidemic was not reflected in the total mononuclear leucocyte count of the seal pups, but they had an absolute granulocytosis, thrombocytosis, anaemia, and high total white blood cell counts. When first examined, 11 of the pups had a positive serum IgG titre, and four had a positive serum IgM titre. High levels of PDV-specific serum IgG antibodies were not correlated with an absence of clinical signs or longer survival.

Quantitative analysis of the 2002 phocine distemper epidemic in the Netherlands.

Phocine distemper virus (PDV) caused thousands of deaths among harbor seals (Phoca vitulina) from the North Sea in 1988 and 2002. To examine the effects of different factors on the pathology of phocine distemper, we performed necropsies and laboratory analyses on 369 harbor seals that stranded along the Dutch coast during the 2002 PDV epidemic. Diagnostic tests for morbillivirus infection indicated a differential temporal presence of morbillivirus in lung and brain. Seals of 3 years or older were significantly more often IgG positive than younger seals. The most frequent lesions in PDV cases were bronchopneumonia, broncho-interstitial pneumonia, and interstitial emphysema. Extra-thoracic emphysema was rare in <1-year-olds compared with older seals, even though severe pneumonia was more common. PDV cases generally had empty stomachs and less blubber than by-caught seals from before the epidemic. In PDV cases involving older animals, lung, kidney, and adrenal weights were significantly increased. Bordetella bronchiseptica was isolated from lungs in two thirds of the PDV cases examined. Our results indicate that brain should be included among the tissues tested for PDV by RT-PCR; that either phocine distemper has a longer duration in older seals or that there are age-related differences in immunity and organ development; that dehydration could play a role in the course and outcome of phocine distemper; and that bacterial coinfections in lungs are more frequent in PDV cases than gross lesions suggest. These results illustrate how quantitative analysis of pathology data from such epidemics can improve understanding of the causative disease.

The development of a qualitative real-time RT-PCR assay for the detection of hepatitis C virus.

Real-time polymerase chain reaction (PCR) represents a favourable option for the detection of hepatitis C virus (HCV). A real-time reverse transcriptase PCR (RT-PCR) assay was developed as a qualitative diagnostic screening method for the detection of HCV using the ABI PRISM 7500 Sequence Detection System. The primers and probe were designed to target the 5'-untranslated region of the hepatitis C viral genome. A second heterologous probe assay was developed for the detection of the haemagglutinin gene of phocine distemper virus (PDV) and was used as an internal control. A semi-automated HCV extraction method was also implemented using the ABI PRISM 6100 Nucleic Acid PrepStation. The HCV assay was optimised as a qualitative singleplex RT-PCR assay with parallel testing of the target and internal control. The assay results (n = 200) were compared to the COBAS AMPLICOR HCV Test v2.0 assay. The assay demonstrated a high rate of sensitivity (99%), specificity (100%) and an acceptable limit of detection (LOD) of 100 IU/ml. The development of a qualitative multiplex assay for the simultaneous detection of HCV and internal control indicates the same high rates of sensitivity and specificity. This sensitive real-time assay may prove to be a valuable method for the detection of HCV.

Continuum description of a contact infection spread in a SIR model.

We consider the process of an epidemic spread in a population of individuals with low mobility within the SIR scheme. In a continuum limit such propagation mechanism is described by a non-linear reaction-diffusion equation with a diffusion coefficient being the function of the densities of susceptible. The traveling wave solution of the corresponding system of partial differential equations is obtained and analyzed. We show that the model allows for description of Kendall epidemic waves, and give the dependence of the infection wave's shape on the parameters of the system. An explicit calculation is done for realistic values of parameters obtained from field epidemiological data for a phocine distemper virus infection among harbor seals in 1988.

Identification and real-time PCR quantification of Phocine distemper virus from two colonies of Scottish grey seals in 2002.

The North Sea European harbour seal (Phoca vitulina) population has endured two phocine distemper virus (PDV) epidemics in 1988 and 2002. The grey seal (Halichoerus grypus) is a sympatric seal species that shows little or no mortality from PDV. Two Scottish grey seal breeding colonies were sampled for evidence of PDV infection approximately 2 months after the peak of the 2002 epidemic. In both colonies, a proportion of mothers (13/109) and pups (6/84) tested positive for PDV in their leukocytes. All infected animals were asymptomatic and completed the breeding season successfully. These results illustrate that grey seals come into contact with infectious seals and can become infected themselves without experiencing acute effects. In some seals the virus is able to replicate from the primary site of infection. This study provides evidence that grey seals may have an active role in the spread of PDV during an epidemic.

Phocine distemper in German seals, 2002.

Approximately 21,700 seals died during a morbillivirus epidemic in northwestern Europe in 2002. Phocine distemper virus 1 was isolated from seals in German waters. The sequence of the P gene showed 97% identity with the Dutch virus isolated in 1988. There was 100% identity with the Dutch isolate from 2002 and a single nucleotide mismatch with the Danish isolate.

Retrospective differentiation of canine distemper virus and phocine distemper virus in phocids.

Formalin-fixed paraffin-embedded tissues from one Caspian seal (Phoca caspica), one harp seal (Phoca groenlandica), one hooded seal (Cystophora cristata), and one harbor seal (Phoca vitulina vitulina) were used to compare the utility of immunohistochemistry (IHC) versus that of a novel seminested reverse transcriptase polymerase chain reaction (RT-PCR) to detect and differentiate canine distemper virus (CDV) and phocine distemper virus (PDV). Four antibodies made against PDV were able to detect both viruses. Two antibodies made against cetacean morbillivirus (CMV) did not label antigens from either CDV or PDV. A third anti-CMV antibody inconsistently stained CDV antigens but did not label PDV antigens. The seminested RT-PCR was able to detect RNA of the phosphoprotein gene in all positive cases. Nucleotide sequence analyses of seminested RT-PCR products were used to differentiate CDV RNA from PDV RNA. From these data, it was determined that IHC using antibodies generated against PDV provided a rapid means of detection for both CDV and PDV antigens; however, differentiation between CDV and PDV was achieved only with the RT-PCR assay.

Nucleotide sequence analysis of the large (L) genes of phocine distemper virus and canine distemper virus (corrected sequence).

This paper corrects the previously published sequence of the L gene of canine distemper virus (CDV). Errors in the published sequence (M. S. Sidhu et al., 1993, Virology 193, 50-65) led to frame shifts between residues 1021-1032, 1190-1219 and 1645-1650; a deletion of 21 amino acids between residues 1684-1705, and a single residue deletion at residue 1478. Residue 237 is now found to be glycine rather than tryptophan and residue 1626 proline instead of threonine. The sequence of the L gene of phocine distemper virus (PDV) was also determined. Alignment of the morbillivirus L proteins showed that PDV and CDV are more closely related to each other than to rinderpest virus and measles virus. Two regions of low identity are proposed to function as hinge regions between three highly conserved domains (I-III) in the morbillivirus L proteins. New sequence motifs have been identified on the basis of conservation in the morbilliviruses and the Paramyxovirinae.

Morbilliviruses and morbillivirus diseases of marine mammals.

In recent years, serious disease outbreaks among seals and dolphins were attributed to infection with established or newly recognized morbilliviruses. The first identification of a morbillivirus as causative agent of mass mortality among marine mammals was in 1988, when the previously unrecognized phocine distemper virus (PDV) caused the death of 20,000 harbor seals (Phoca vitulina) in northwestern Europe. A similar epizootic among Baikal seals (Phoca sibirica) in Siberia in 1987 was later attributed to infection with canine distemper virus (CDV). A morbillivirus isolated from stranded harbor porpoises (Phocoena phocoena) between 1988 and 1990 proved to be yet another new member of the genus Morbillivirus, distinct from PDV and CDV and more closely related to rinderpest virus and peste-des-petits-ruminants virus: porpoise morbillivirus. A similar virus, dolphin morbillivirus, was the primary cause of mass mortality among striped dolphins (Stenella coeruleoalba) in the Mediterranean from 1990 to 1992. In this review, current knowledge of the genetic and antigenic relationships of these viruses is presented, and the origin and epizootiological aspects of the newly discovered morbilliviruses are discussed. In addition, the possible contributory role of environmental contaminant-related immunosuppression in the severity and extent of the different disease outbreaks is discussed.

Characterisation of morbilliviruses isolated from Lake Baikal seals (Phoca sibirica).

Sequence analysis of the haemagglutinin protein (H) gene of the morbillivirus (PDV-2) isolated from a Siberian seal (Phoca sibirica) during the 1987/1988 epizootic in Lake Baikal revealed that it was most closely related to two recent isolates of canine distemper virus (CDV) from Germany and different from CDV vaccines currently in use in that region. The virus continued to circulate in seals in Lake Baikal after the 1987/1988 epizootic since sera collected from culled seals in the spring of 1992 were positive in morbillivirus ELISA tests, reacting most strongly with the CDV antigen.